This invention relates to polyamide-alkylator conjugates and related products, including methods of using such conjugates as well as methods relating to their synthesis.
None of the following discussion of the background of the invention, which is provided solely to aid the reader in understanding the invention, is admitted to be or to describe prior art to the invention.
The design of synthetic ligands capable of reading information stored in the DNA double helix has been a long-standing goal of chemistry and molecular biology. Cell-permeable small molecules, which target predetermined nucleotide sequences in double-stranded nucleic acids, particularly double-stranded DNA (dsDNA), are useful in regulating, or modulating, gene-expression. Small molecules specifically targeted to any predetermined DNA sequence would be useful tools in molecular biology and potentially in human medicine.
Oligodeoxynucleotides that recognize the major groove of double-helical DNA via triple-helix formation bind to a broad range of sequences with high affinity and specificity. See Moser, et al., Science, vol. 238:645-650 (1987), Duvaivalentin, et al., Proc. Nat""l Acad. Sci. USA, vol. 89:504-508 (1992), Maher, et al., Biochemistry, vol. 31:70-81 (1992). Although oligonucleotides and their analogs have been shown to interfere with gene expression, the triple helix approach so far has been limited to purine tracks and suffers from poor cellular uptake.
Another recent approach to targeting specific nucleotide sequences in dsDNA has involved molecules known as xe2x80x9cpolyamides.xe2x80x9d See U.S. Ser. No. 08/607,078, PCT/US97/03332, U.S. Ser. Nos. 08/837,524, 08/853,525, PCT/US97/12733, U.S. Ser. No. 08/853,522, PCT/US97/12722, PCT/US98/06997, PCT/US98/02444, PCT/US98/02684, PCT/US98/01006, PCT/US98/03829, and PCT/US98/0714 all of which are incorporated herein by reference in their entirety, including any drawings. As described in the foregoing references, polyamides comprise polymers of amino acids covalently linked by amide bonds. Preferably, the amino acids used to form these polymers include N-methylpyrrole (Py) and N-methylimidazole (Im). Polyamides containing pyrrole (Py), and imidazole (Im) amino acids are synthetic ligands that have an affinity and specificity for DNA comparable to naturally occurring DNA binding proteins Trauger, J. W., Baird, E. E. and Dervan, P. B. (1996), Nature 382, 559-561; Swalley, S. E., Baird, E. E. and Dervan, P. B. (1997), J. Am. Chem. Soc. 119, 6953-6961; Turner, J. M., Baird, E. E. and Dervan, P. B. (1997), J. Am. Chem. Soc. 119, 7636-7644; Trauger, J. W., Baird, E. E. and Dervan, P. B. (1998), Angewandte Chemie-International Edition 37, 1421-1423; and Dervan, P. B. and Burli, R. W. (1999), Current Opinion in Chemical Biology 3, 688-693.
The particular order of amino acids in such polyamides, and their pairing in dimeric, antiparallel complexes formed by association of two polyamide polymers, determines the sequence of nucleotides in dsDNA with which the polymers preferably associate. The development of pairing rules for minor groove binding polyamides derived from N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids provided a useful code to control target nucleotide base pair sequence specificity. Specifically, an Im/Py pair in adjacent polymers was found to distinguish Gxe2x80xa2C from Cxe2x80xa2G and both of these from Axe2x80xa2T or Txe2x80xa2A base pairs. A Py/Py pair was found to specify Axe2x80xa2T from Gxe2x80xa2C but could not distinguish Axe2x80xa2T from Txe2x80xa2A. More recently, it has been discovered that inclusion of a new aromatic amino acid, 3-hydroxy-N-methylpyrrole (Hp) (made by replacing a single hydrogen atom in Py with a hydroxy group), in a polyamide and paired opposite Py enables Axe2x80xa2T to be discriminated from Txe2x80xa2A by an order of magnitude. Utilizing Hp together with Py and Im in polyamides provides a code to distinguish all four Watson-Crick base pairs (i.e.. Axe2x80xa2T, Txe2x80xa2A, Gxe2x80xa2C, and Cxe2x80xa2G) in the minor groove of dsDNA, as described in Table 1.
As discussed above, a number of different polyamide motifs have been reported in the literature, including xe2x80x9chairpins,xe2x80x9d xe2x80x9cH-pins,xe2x80x9d xe2x80x9coverlapped,xe2x80x9d xe2x80x9cslipped,xe2x80x9d and xe2x80x9cextendedxe2x80x9d polyamide motifs. Specifically, hairpin polyamides are those wherein the carboxy terminus of one amino acid polymer is linked via a linker molecule, typically aminobutyric acid or a derivative thereof to the amino terminus of the second polymer portion of the polyamide. Indeed, the linker amino acid xcex3-aminobutyric acid (xcex3), when used to connect first and second polyamide polymer portions, or polyamide subunits, Cxe2x86x92N in a xe2x80x9chairpin motif,xe2x80x9d enables construction of polyamides that bind to predetermined target sites in dsDNA with more than 100-fold enhanced affinity relative to unlinked polyamide subunits. See, for example, Turner, et al., J. Am. Chem. Soc., vol. 119:7636-7644 (1997), Trauger, et al., Angew. Chemie. Int. Ed. Eng., vol. 37: 1421-1423 (1997), Turner, et al., J. Am. Chem. Soc., vol. 120:6219-6226 (1998), and Trauger, et al., J. Am. Chem. Soc., vol. 120:3534-3535 (1998). Paired xcex2-alanine residues (xcex2/xcex2), restore the curvature of the dimer for recognition of larger binding sites and in addition, code for AT/TA base pairs Trauger, J. W., Baird, E. E., Mrksich, M. and Dervan, P. B. (1996), J. Am. Chem. Soc. 118, 6160-6166; Swalley, S. E., Baird, E. E. and Dervan, P. B. (1997), Chem.-Eur. J. 3, 1600-1607; and Trauger, J. W., Baird, E. E. and Dervan, P. B. (1998), J. Am. Chem. Soc. 120, 3534-3535. Eight ring hairpin polyamides can bind a 6 base pair match sequence at subnanomolar concentrations with good sensitivity to mismatch sequences. Dervan, P. B. et al. Curr. Opin. Chem. Biol. 1999, 3,688-693. Moreover, eight-ring hairpin polyamides (comprised of two four amino acid polymer portions linked Cxe2x86x92N) have been found to regulate transcription and permeate a variety of cell types in culture. See Gottesfield, J. M.; et al., Nature, vol. 387:202-205 (1997).
An H-pin polyamide motif, i.e., wherein two paired, antiparallel polyamide subunits are linked by a linker covalently attached to an internal polyamide pair, have also been reported. Another polyamide motif that can be formed between linked or unlinked polyamide subunits is an xe2x80x9cextendedxe2x80x9d motif, wherein one of the polyamide subunits comprises more amino acids than the other, and thus has a single-stranded region. See U.S. Ser. No. 08/607,078. In contrast, an xe2x80x9coverlappedxe2x80x9d polyamide is one wherein the antiparallel polyamide subunits completely overlap, whereas in a xe2x80x9cslippedxe2x80x9d binding motif, the two subunits overlap only partially, with the C-terminal portions not associating with the N-terminal regions of the other subunit. See U.S. Ser. No. 08/607,078.
DNA alkylation by Duocarmycin A segmentxe2x80x94hairpin polyamide conjugates is described in Tao et al., J. Am. Chem. Soc. 1999, 121:4961-4967. Alkylation by one such conjugate was reported to proceed with efficiency at nanomolar concentration against a particular DNA sequence. Other Duocarmycin A experiments we reported in Fujiwara, et al. J. Am. Chem. Soc. 1999, 121:7706-7702 and Tao, et al. Agnew. Chem. Int. Ed. 1999, 38; No. 5, pages 650-653.
The present invention is based on the surprising and unexpected discovery of new and useful polyamide-alkylator conjugates. As a result of their DNA binding properties, polyamides deliver reactive moieties for covalent reaction at specific DNA sequences and thereby inhibit DNA-protein interactions. This site specific alkylation of DNA is a useful tool to regulate gene expression. In addition to competing with transcription factors or promoters, the conjugates of the present invention will be used to target a gene""s coding region. This will allow use of synthetic chemistry to create a new class of gene specific xe2x80x9cknockoutxe2x80x9d reagents which will be useful in biological disciplines.
We have designed and synthesized a class of hairpin polyamides equipped with DNA alkylating agents and characterized the specificity and yield of covalent modification. In one instance Bis(dichloroethylamino)benzene derivatives of the well-characterized alkylator chlorambucil (CHL) were attached to the xcex3-turn of an eight ring hairpin polyamide targeted to the HIV promoter. The hairpin polyamide-CHL conjugate binds and selectively alkylates predetermined sites in the HIV promoter at subnanomolar concentrations. Cleavage sites were determined on both strands of a restriction fragment containing the HIV-1 promoter revealing good specificity and high yield of alkylation. The ability of polyamide-CHL conjugates to sequence specifically alkylate double stranded DNA in high yield and at low concentration indicates their use as regulators of gene expression in cell culture and complex organisms.
In another instance, we synthesised and characterized an eight-ring hairpin polyamide conjugated at the xcex3-turn to both enantiomers of 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI), an alkylating moiety related to CC-1065. We attached the seco-CBI unit to the hairpin turn. In addition, we used the two different diastereomers which afforded opposite strand reactivity in the minor groove depending on mirror image (FIG. 11) (SEQ ID NO:17). Alkylation yield and specificity were determined on a restriction fragment containing six base pair match and mismatch sites. Alkylation was observed at a single adenine flanking the polyamide binding site, and strand selective cleavage could be achieved based on the enantiomer of seco-CBI chosen. At 1 nM concentrations of polyamide-seco-CBI conjugate, near quantitative cleavage was observed after 12 hours. Thus, these bifunctional molecules are useful for targeting coding regions of genes and inhibition of transcription.
Thus, in one aspect, the invention provides a polyamide-alkylator conjugate. The conjugate contains an alkylator linked to a polyamide. The polyamide contains at least one pyrrole or imidazole amino acid and may bind DNA with subnanomolar binding affinity. The alkylator is linked to the y-aminobutyric acid (or other turn moiety) of a hairpin polyamide or the alkylator selectively alkylates only one strand of a double-stranded DNA. By xe2x80x9cturn moietyxe2x80x9d is meant a chemical group that operates to orient two strands or segments of a polyamide substantially parallel to one another. Preferred turn moieties are those which comprise an aliphatic chain, for example, aminobutyric acid, with the latter being particularly preferred. Particularly preferred aminobutyric acids include xcex3-aminobutyric acid, particularly substituted derivatives thereof, for example, (R)-2,4-diaminobutyric acid. Preferably, the conjugate of the invention has separate domains for DNA binding (e.g., the polyamide region of the conjugate) and DNA covalent attachment (e.g., the alkylation region of the conjugate).
The term xe2x80x9cpolyamidexe2x80x9d is used to describe a minor groove-targeting polypeptide, which is a polymer of amino acids chemically bound by amide linkages (CONH). An xe2x80x9camino acidxe2x80x9d is defined as an organic molecule containing both an amino group (NH2) and a carboxylic acid (COOH). The polyamides of this invention may be comprised of imidazole carboxamides, pyrrole carboxamides, aliphatic amino acids, aromatic amino acids and any chemical modifications thereof. In preferred embodiments, the polyamide is a hairpin polyamide, for example a hairpin polyamide that contains eight or more heterocyclic rings, wherein each of the eight heterocylic rings is a pyrrole ring or an imidazole ring. The polyamide preferably binds DNA with subnanomolar binding affinity, which is measured as described in examples herein.
As used herein xe2x80x9cpolyamidexe2x80x9d preferably refers to a polymer derived from subunits chosen from the set below, in the form of the corresponding carboxamides: 
where R1 is C1-100 alkyl (preferably C1-10 alkyl such as methyl, ethyl, isopropyl), C1-100 alkylamine (preferably C1-10 alkylamine such as ethylamine), C1-100 alkyldiamine (preferably C1-10 alkyldiamine such as N,N-dimethylpropylamine), a C1-100 alkylcarboxylate (preferably a C1-10 alkylcarboxylate such as CH2COOH), C1-100 alkenyl (preferably C1-10 alkenyl such as CH2CHxe2x95x90CH2), or a C1-100 alkynyl (preferably C1-10 alkenyl such as CH2CHxe2x89xa1CH3), or a C1-100L, where L groups can be independently chosen from biotin, oligodeoxynucleotide, N-ethylnitrosourea, fluorescein, bromoacetamide. iodoacetamide, DL-xcex1-lipoic acid, acridine, ethyl red, 4-(psoraen-8-yloxy)-butyrate, tartaric acid, (+)-xcex1-tocopheral;
where R2 and 2R are independently chosen from H, NH2, OH, SH, Cl, Br, F, N-acetyl, or N-formyl;
where R3 is H, NH2, OH, SH, Br, Cl, F, OMe, CH2OH, CH2SH, CH2NH2; and
where X is chosen from N, CH, COH, CCH3, CNH2, CCl, CF.
More preferably, xe2x80x9cpolyamidexe2x80x9d refers to a polymer of comprising one or more subunits of the formula (II), below 
where R1 is chosen from H, NH2, SH, Cl, Br, F, N-acetyl, or N-formyl;
where R2 is C1-100 alkyl (preferably C1-10 alkyl such as methyl, ethyl, isopropyl), C1-100 alkylamine (preferably C1-10 alkylamine such as ethylamine), C1-100 alkyldiamine (preferably C1-10 alkyldiamine such as N,N-dimethylpropylamine), a C1-100 alkylcarboxylate (preferably a C1-10 alkylcarboxylate such as CH2COOH), C1-100 alkenyl (preferably C1-10 alkenyl such as CH2CHxe2x95x90CH2), or a C1-100 alkynyl (preferably C1-10 alkynyl such as xe2x80x94C2Cxe2x89xa1CH3), or a C1-100L, where L groups can be independently chose from biotin, oligodeoxynucleotide, N-ethylnitrosourea, fluorescein, bromoacetamide, iodoacetamide, DL-xcex1-lipoic acid, acridine, ethyl red, 4-(psoraen-8-yloxy)-butyrate, tartaric acid, (+)-xcex1-tocopherol;
where R3 is chosen from H, NH2, OH, SH, Br, Cl, F, Ome, CH2OH, CH2SH, CH2NH2;
where R4 is xe2x80x94NH (CH2)0-100NR5R6 or NH(CH2)mCO NH(CH2)0-100 NR5R6 or NHR5 or NH(CH2)mCONHR5; Where R5 and R6 are independently chosen from H, Cl, NO, N-acetyl, benzyl, C1-100 alkyl, C1-100 alkylamine, C1-100 alkyldiamine, C1-100 alkylcarboxylate, C1-100 alkenyl, a C1-100 alkynyl; where m is an integer value ranging from 0 to 12;
where X and Y are chosen from the following, N, CH, COH, CCH3, CNH2, CCl, CF;
a is an integer chosen from values of 0 or 1
b is an integer chosen integer values ranging from 1 to 5; and
c is an integer value ranging from 2 to 10.
Hereinafter, N-methylpyrrolecarboxamide may be referred to as xe2x80x9cPyxe2x80x9d, N-methylimidazolecarboxamide may be referred to as xe2x80x9cImxe2x80x9d, xcex3-aminobutyric acid may be referred to as xe2x80x9cxcex3xe2x80x9d, xcex2-alanine may be referred to as xe2x80x9cxcex2xe2x80x9d, glycine may be referred to as xe2x80x9cGxe2x80x9d, dimethylaminopropylamide may be referred to as xe2x80x9cDpxe2x80x9d, and ethylenediaminetetraacetic acid may be referred to as xe2x80x9cEDTAxe2x80x9d.
By xe2x80x9calkylatorxe2x80x9d is meant a compound that reacts with and adds an alkyl group to another molecule. In preferred embodiments, the alkylator is reactive with DNA at about 37 degrees celsius, the alkylator is substantially inert in aqueous media, and/or the conjugate is present in a buffer and the alkylator is non-reactive with the buffer. The alkylator may be cyclophosphamide, nitrosoureas, mitozolomide, anthramycin, bromoacetyl, a nitrogen mustard, a derivative of chlorambucil (such as a Bis(dichloroethylamino)benzene derivative), seco-CBI, mitomycin, initomycin C, or (+)-CC- 1065. Seco-CBI is a precursor to 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI), [Boger, D. L. et al. Bioorgan. Med. Chem. 1995, 3, 1429-1453; and Boger, D. L. and Johnson, D. S. Angew. Chem., Int. Ed. Engl.1996, 35, 1438-1474] an analogue of the natural product (+)-CC-1065. CBI shows increased reactivity to DNA as well as increased stability to solvolysis. [Boger, D. L. and Munk, S. A. J. Am. Chem. Soc. 1992, 114, 5487-5496.] The seco agents readily close to the cyclopropane forms and have equivalent reactivity as compared to CBI, but have been shown to have longer shelf lives. [Boger, D. L. et al. Bioorg. Med. Chem. Lett. 1991, 1, 55-58.]
By xe2x80x9clinkedxe2x80x9d is meant that there is a chemical connection between the polyamide and the alkylator. Preferably the linkage is a covalent bond, but the term linked also encompasses non-convalent interactions such as hydrogen bonding, Van der Waals interactions, hydrophobic interactions, and ionic bonding.
In certain preferred embodiments, the alkylator is linked (e.g., covalently linked) to the xcex3-aminobutyric acid of the polyamide (in particular, the alkylator may be covalently attached to a chiral xcex1-amino group on the xcex3-aminobutyric acid of the polyamide), while in other preferred embodiments the alkylator selectively alkylates only one strand of a double-stranded DNA (i.e., only the 5xe2x80x2 strand or only the 3xe2x80x2 strand). For example, the alkylator may selectively alkylate only one strand of a double-stranded DNA when the polyamide portion of the conjugate interacts with the DNA (e.g., is covalently bound to the DNA). As noted above, in other preferred embodiments the polyamide binds to DNA with subnanomolar binding affinity.
As used herein, xe2x80x9csubnanomolar affinityxe2x80x9d means binding that is characterized by a dissociation constant, Kd, of less than 1 nM, as measured by DNase I footprint titration. Preferably, polyamides and/or conjugates of the present invention are characterized by subnanomolar binding affinity for the identified target DNA sequence. As used herein, the xe2x80x9cselectivityxe2x80x9d of the binding of a polyamide or conjugate to a DNA sequence is the ratio of the dissociation constant, Kd, as measured by DNase I footprint titration of binding the polyamide or conjugate to a mismatch DNA sequence divided by the corresponding dissociation constant of the binding of the polyamide or conjugate to the identified target DNA sequence. Preferably, polyamides and/or conjugates of the present invention are characterized by a selectivity of about 5 or greater, more preferably a selectivity of greater that about 10. Of course any combination of the above embodiment is possible, i.e., the alkylator may selectively alkylate only one strand of dsDNA and the polyamide may bind DNA with subnanomolar binding affinity.
The conjugate preferably is capable of sequence specific alkylation of DNA, including sequence specific alkylation of DNA in the minor groove. Thus, the conjugate can be designed to target a predetermined DNA sequence. The conjugate preferably alkylates an adenine adjacent to the binding site and/or has sub-nanomolar binding affinity for the DNA. Preferably, the conjugate is also selective, for example the conjugate has at least 20-fold (more preferably about 100-fold) greater affinity for a target site than for a site differing from the target site by two amino acids. Preferably, a conjugate will interact with its target nucleotide base pair sequence with an affinity, as measured by DNase footprint titration, of less than about 100 nM, preferably less than about 10 nM, more preferably less than about 1.0 nM, even more preferably less than about 0.1 nM. Thus, the conjugates of the invention have substantially the same binding affinity and specificity as the polyamide.
The rate of alkylation is an important measure of polyamide-alkylator ,conjugates. The conjugates of the invention preferably alkylate the DNA at a rate whereby alkylation is at least half completed at one or more match sites in about 2.2 hours, more preferably alkylate the DNA at a rate whereby final cleavage yield on the bottom strand of the DNA is about 45% and even more preferably alkylate the DNA at a rate whereby final cleavage yield on the bottom strand of the DNA is about 96%.
In another aspect, the invention provides a composition containing a conjugate of the invention in a pharmaceutically acceptable carrier. A method of making the composition of the invention is also provided and involves the step of providing a conjugate of the invention in a pharmaceutically acceptable carrier. Pharmaceutical formulations of the invention are described in detail in a section below.
In yet another aspect, the invention features a method of using a conjugate or composition of the invention to deliver a reactive moiety for covalent reaction at one or more DNA sequences. The method involves the step of administering the conjugate or the composition to an organism and thereby modulates DNA-protein interactions and/or gene expression comprising. In a preferred embodiment, the DNA is in the coding region of a gene. Again, detailed procedures for administration are provided in a separate section below. Administration preferably is of a therapeutically effective amount of the agent being administered.
A xe2x80x9ctherapeutically effectivexe2x80x9d amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein, A xe2x80x9cthereapeutically effective amount,xe2x80x9d in reference to the treatment of a cancer refers to an amount sufficient to bring about one or more of the following results: reduce the size of the cancer, inhibit the metastasis of the cancer, inhibit the growth of the cancer, stop the growth of the cancer, relieve discomfort due to the cancer, or prolong the life of a patient inflicted with the cancer. A xe2x80x9ctherapeutically effective amountxe2x80x9d, in reference to the treatment of a cell proliferative disorder other than a cancer refers to an amount sufficient to bring about one or more of the following results: inhibit the growth cells causing the disorder, relieve discomfort due to the disorder, or prolong the life of a patient suffering from the disorder.
The invention also features a method of treating an organism by using two or more conjugates of the invention to selectively remove undesired gene segments or integrated viral DNA""s from a host genome in vivo. The term xe2x80x9ctreatingxe2x80x9d refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism. The term xe2x80x9ctreatingxe2x80x9d preferably refers to ameliorating a symptom of the abnormal condition in a group of patients to whom the conjugate is administered relative to a control group that does not receive the conjugate. The effect of the treatment can be monitored by measuring a change or an absence of a change in cell phenotype, a change or an absence of a change in cell proliferation. The term xe2x80x9ctreatingxe2x80x9d or xe2x80x9ctreatmentxe2x80x9d does not necessarily mean total cure. Any alleviation of any undesired symptom of the disease to any extent or the slowing down of the progress of the disease can be considered treatment. Furthermore, treatment may include acts which may worsen the patient""s overall feeling of well being or appearance. For example, the administration of chemotherapy in cancer patients which may leave the patients feeling xe2x80x9csickerxe2x80x9d is still considered treatment. The method of treatment involves the steps of administering two or more conjugates of the invention to the organism having undesired gene segments or integrated viral DNA""s and thereby selectively removing the undesired gene segments or the integrated viral DNA""s. The conjugates preferably are selected so that one alkylates the initial amino acid of the sequence to be removed and the other conjugate targets the terminal amino acid of such a sequence. The term xe2x80x9corganismxe2x80x9d relates to any living entity comprised of at least one cell. An organism can be as simple as one eukaryotic cell or as complex as a mammal. The organism is preferably a mammal, more preferably a human. The term xe2x80x9cmammalxe2x80x9d refers preferably to such organisms as mice, rats, rabbits, guinea pigs, sheep, and goats, more preferably to cats, dogs, monkeys, and apes.
In another aspect, the invention features a method of making a conjugate of the invention. The method involves the steps of coupling the alkylator to the polyamide under conditions whereby the polyamide links to the alkylator and forms the polyamide-alkylator conjugate. Detailed procedures for making preferred conjugates of the invention are provided in the examples below and those skilled in the art would be able to make other conjugates of the invention based on these examples. A hairpin polyamide-bromoacrylic acid conjugate can be synthesized in an analogous manner. The free amino of the (R)-2,4 diaminobutyric acid in the xcex3-turn position of the polyamide can be coupled to bromoacrylic acid with EDC as the coupling agent.
Other aspects of the invention feature a cell containing a conjugate of the invention and a method of making such a cell by providing the conjugate to a cell under conditions whereby the conjugate enters the cell. Also featured is a method of using such a cell which involves the step of administering a test compound to the cell and measuring the effect of the test compound on the cell. Another method of using such a cell is in a method of treating an organism which involves the step of administering the cell to an organism in need of such treatment.
Other aspects relate to methods of using the conjugates of the invention. One area of application relates to the modulation of gene expression. xe2x80x9cModulationxe2x80x9d refers to activating, increasing, enhancing, derepressing, reducing, decreasing, inhibiting, or preventing expression of a gene. Thus, some conjugatess positively affect, or up-regulate, gene expression, while others negatively affect, or down regulate, gene expression. A xe2x80x9cgenexe2x80x9d refers to genetic locus that encodes one or more gene products. As those in the art will appreciate, a gene can encode more than one gene product by virtue of differential mRNA splicing. Gene products include proteins (e.g., enzymes, receptors, antibodies, growth factors, and hormones) and RNA molecules, particularly tRNAs, ribosomal RNAs and other RNAs which are subunits of multi-component complexes (e.g., telomerase), and catalytic RNAs (e.g., ribozymes). To achieve the desired level of modulation in systems comprising cells, it is necessary to deliver a sufficient quantity of a conjugate according to the invention to the cells.
In some embodiments, the use of conjugates according to the invention can modulate the expression of more than one gene. For example, more than one conjugate, each of which specifically modulates a particular gene, can be delivered. Alternatively, the conjugate may directly influence the expression of more than one gene. For example, if the conjugate targets a nucleotide base pair sequence found in a regulatory region of more than one gene, modulation of expression of multiple genes may occur. Alternatively, the conjugate may exhibit its expression-modulating effects indirectly, or by a combination of direct and indirect effects. For instance, if the conjugate inhibits expression of a phosphatase that removes phosphates from a plurality of proteins, the expression of genes regulated by pathways that involve the phosphatase will be affected.
Certain embodiments of this aspect concern modulation of gene expression in vitro. xe2x80x9cIn vitroxe2x80x9d includes both in situ and cell-free environments (e.g., a cell extract or in a well-defined reaction medium). Thus, the conjugatess of the invention can be used to modulate gene expression in cultured cells, such as may be used in ex vivo therapy or research.
Other embodiments of this aspect relate to the modulation of gene expression in vivo, some of which concern therapeutic purposes. As used herein, a xe2x80x9ctherapeutic purposexe2x80x9d includes both therapy (i.e., treatment of an existing condition) and prophylaxis (i.e., prevention). Representative examples of a therapeutic purpose include treatment of a disease associated with aberrant expression of the gene of interest (as occurs in certain cancers and genetic diseases, for example), as well as treatment of a disease associated with the presence of a pathogen (a virus or a bacterial or eukaryotic pathogenic organism).
Conjugates according to the invention can be used for therapeutic purposes in conjunction with a vast array of organisms, including both animals and plants. Preferred animals amenable to application of the therapeutic and prophylactic methods herein described include animals of agricultural importance, for example, avian (particularly poultry), bovine, equine, ovine, and porcine animals, companion animals such as dogs and cats, and humans. With regard to plants, preferred plants include those of agricultural importance, including cereals, grains, and grasses. Similarly, conjugatess according to the invention can be developed to control pests, e.g., certain insects and rodents.
The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.